Cancer treatment including glycolytic inhibitors

ABSTRACT

Glycolytic inhibitors are useful in the treatment of solid tumors by attacking anaerobic cells at the center on the tumor. 2-deoxyglucose, oxamate and various analogs thereof are identified as having a natural selective toxicity toward anaerobic cells, and will significantly increase the efficacy of standard cancer chemotherapeutic and radiation regiments as well as new protocols emerging with anti-angiogenic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 10/953,088 filed Sep. 29,2004 which was a divisional of application Ser. No. 10/401,457 filedMar. 28, 2003 now U.S. Pat. No. 7,160,865, which was a divisional ofapplication Ser. No. 09/561,720 filed May 1, 2000, now U.S. Pat. No.6,670,330, which are herein incorporated by reference.

STATEMENT OF GOVERNMENT INTEREST

The United States Federal Government may have certain rights with regardto this invention under NIH grant CA-37109-10.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates, in general, to compositions and methodsaimed at effectively treating anaerobic tumor cells with inhibitors ofglycolysis. It also extends to novel and useful methods and compositionsfor treating aerobic tumor cells with inhibitors of oxidativephosphorylation in combination with glycolytic inhibitors. Inhibitingoxidative phosphorylation to convert aerobic tumor cells to anaerobiccells, hypersensitizes them to glycolytic inhibitors.

U.S. patents that are relevant to the compositions and methods of thepresent invention are: U.S. Pat. Nos. 4,840,939 and 4,684,627, both fora treatment of cancer with phlorizin and its derivatives; U.S. Pat. No.4,683,222 for N-glycosylated carboxamide derivatives and their use forinfluencing the body's inherent defenses; and U.S. Pat. No. 4,420,489for sugars with sulfur replacing the ring oxygen atom as antiradiationagents. U.S. Pat. No. 4,840,939 is also relevant for its discussion ofthe work of Warburg in 1931, concerning the way cancer cells metabolizeglucose.

Cancer cells at the inner core of a tumor are poorly oxygenated andconsequently relyon anaerobic metabolism for survival. In this conditiontumor cells divide more slowly than outer growing aerobic cells andconsequently are more resistant to standard chemotherapeutic agentswhich target rapidly dividing cells. Thus, cells growing anaerobicallyin these instances exhibit a form of multidrug resistance (MDR) whichcontributes to chemotherapy failures in the treatment of solid tumors.Anaerobiosis, however, also provides a natural window of selectivity foragents that interfere with glycolysis.

This realization forms the basis for the present invention. According tothe present invention, new opportunities are provided for increasing theefficacy of chemotherapeutic protocols. With data and knowledgeaccumulated by us in our previous work on mitochondrial agents, and withadditional work performed by us now, various hypotheses have beenformulated and verified to prove the efficacy of the present inventionwith regard to its compositions and its methods.

See, for example, a series of papers coauthored by one of the presentinventors: Lampidis, T J, Bernal, S D, Summerhayes, I C, and Chen, L B.“Rhodamine 123 is selectively toxic and preferentially retained incarcinoma cells in vitro.” NY Acad. Sci. 397:299-302, 1982; Summerhayes,I C, Lampidis, T J, Bernal, S D, Shepherd, E L, and Chen, L B. “Unusualretention of Rhodamine 123 by mitochondria in muscle and carcinomacells.” Proc. Natl. Acad. Sci. USA 79:5292-5296, 1982; Bernal, S B,Lampidis, T J, Summerhayes, I C, and Chen, L B. “Rhodamine 123selectively reduces clonogenic ability of carcinoma cells in vitro.”Science. 218:1117-1119, 1982; Lampidis, T J, Bernal, S D, Summerhayes, IC, and Chen, L B. “Selective toxicity of Rhodamine 123 in carcinomacells in vitro.” Cancer Res. 43:716-720, 1983; and Bernal, S B,Lampidis, T J, Mclsaac, B, and Chen, L B. “Anticarcinoma activity invivo of Rhodamine 123, a mitochondrial-specific dye.” Science.222:169-172, 1983; which showed that Rhodamine 123 (Rho 123) whichlocalizes in mitochondria of living cells, and uncouples ATP synthesisfrom electron transport, preferentially accumulates in, and kills, avariety of tumor cells as compared to a number of normal cells. Wereasoned for the present invention that tumor cells treated with thisdrug would have to rely solely on glycolysis for ATP production and thusbecome hypersensitized to inhibitors of glycolysis, like 2-dg (2-dg). Incontrast, mitochondrial function in normal cells remained unaffectedwhen treated with Rho 123 and therefore these cells were nothypersensitive to 2-dg. In fact, we found that co-treating human breastcarcinoma cells, MCF-7, with Rho 123 and 2-dg, at a dose of Rho 123 thatalone inhibited 50% of colony forming units, and at a dose of 2-dg whichproduced no toxicity, 100% of the colony forming units was inhibited.

This concept was carried over to in vivo studies in which it was foundthat animals with implanted tumors that were treated in combination with2-dg and Rho 123 were cured whereas when treated with either drug alone,only partial or no responses were obtained. This latter result providesevidence that manipulation of Oxphos and glycolysis simultaneously cancure tumors in animals. Furthermore, this in vivo data also demonstratesthat 2-deoxyglocose can be administered safely to animals, at doseswhich are effective for anti-tumor activity in combination with anOxphos inhibitor. In this regard, several reports have shown that lowlevels of 2-dg can be safely administered to animals for various reasonsincluding hypersensitization of tumors to irradiation.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that glycolyticinhibitors and analogs thereof, are selectively toxic to tumor cellswhich are metabolizing anaerobically. Thus, in conjunction with standardchemotherapy and/or radiation which is focused on aerobic, fast-growingcells, which we will here refer to collectively and individually as“aerobic treatment”, use of these inhibitors will add to the efficacy ofcancer treatment by selectively killing the anaerobically slow-growingtumor cells found at the inner core of solid tumors which are usuallythe most resistant and consequently the most difficult to eradicateusing aerobic treatments.

As an aid for explaining and for demonstrating the usefulness of thepresent invention, we refer to the equation (A)=(B)=(C) where:

(A)=tumor cells treated at a dose of rhodamine 123 which specificallyuncouples ATP synthesis from electron transport;

(B)=ρ^(o) cells which are cells that contain no mitochondrial DNA andtherefore cannot undergo oxidative phosphorylation; and

(C)=tumor cells which are growing anaerobically.

In all three classes, the cells can only produce ATP via theEmbden-Myerhoff pathway (glycolysis) and thus are naturallyhypersensitive to all inhibitors of glycolysis exemplified by2-deoxyglucose, oxamate and novel compounds of the present invention.

Although Warburg originally proposed that tumor cells depend less onmitochondrial function for ATP production and more on glycolysis thannormal cells, there has been no definitive data to date to confirm thishypothesis. There is data however which indicates that cells, tumor ornormal, which are compromised aerobically and switch to anaerobicmetabolism increase their uptake and utilization of glucose. Theincreased uptake has been attributed to the increased appearance ofglucose receptors on the plasma membrane. The fact that cells growing atthe inner core of tumors rely more on glycolysis than cells on the outeredge most likely accounts for the successful use of the radioactiveanalog of glucose, 2-deoxyglucose (2-dg), as a diagnostic tool forlocalizing tumors. This has been the main usage of 2-dg for cancer.Moreover, it has been suggested that 2-dg is taken up more by anaerobiccells due to enhanced expression of glucose receptors. Thus, since acell that is functioning normally relies mainly on oxidativephosphorylation for its supply of ATP when this mechanism is compromisedor absent, glycolysis (the only other way of producing ATP)automatically becomes enhanced. This is precisely why inner core tumorcells should naturally become hypersensitive to agents which blockglycolysis, i.e. 2-dg and others that block the glycolytic pathway atdifferent steps, such as oxamate and iodoacetate.

Oxamate is another agent which blocks glycolysis and is more specificthan 2-dg for anaerobically metabolizing cells. Oxamate has been shownto inhibit lactic dehydrogenase, the enzyme which breaks down pyruvateto lactic acid. Thus, the fact that oxamate blocks glycolysis at adifferent step than 2-dg, and is clearly active in our in vitro cellsystems in selectively killing anaerobic cells (models A and B), lendsfurther proof that our discovery is correct and works according to theprinciples we set forth in this application. The enzyme reaction whichconverts pyruvate to lactate occurs only when the cell's ability toprocess oxygen becomes limited. Thus, aerobically metabolizing cells donot use this enzyme and inhibitors of this reaction will inherently bemore selective than inhibitors of glycolysis such as 2-deoxyglucosesince these latter agents inhibit reactions that occur in aerobic aswell as anaerobic cells.

Iodoacetate, on the other hand, is known to inhibit glycolysis atanother step in the pathway, namely at glyceraldehyde 3-phosphatedehydrogenase, which normally yields 1,3 biphosphoglycerate fromglyceraldehyde 3-phosphate and requires NAD+ to proceed.

The inventors have investigated this overall concept by utilizing anosteosarcoma cell line that has been selected for loss of mtDNA, andthus cannot produce ATP by oxidative phosphorylation (cells (B) above).The inventors have also utilized this concept as a model to determinewhether any test drug utilizes functional mitochondria as a cytotoxictarget. The model indicates that a drug which interferes with ATPproduction in mitochondria of whole cells, but not necessarily inisolated mitochondrial preparations, will hypersensitize a cell toglycolytic inhibitors, i.e. 2-dg, oxamate and iodoacetate. Data has beendeveloped by the inventors which clearly shows that in the cases listedas (A) and (B) above, both are hypersensitive to 2-deoxyglucose, oxamateand iodoacetate which forms the basis for the present invention.

The data which supports the invention that anaerobic cells within atumor are selectively sensitive to glycolytic inhibitors, indicates thatthis is a universal phenomenon and that most or all glycolyticinhibitors will, in general, be similarly effective.

The various features of novelty which characterize the invention arepointed out with particularity in the claims annexed to and forming apart of this disclosure. For a better understanding of the invention,its operating advantages and specific objects attained by its uses,reference is made to the accompanying descriptive matter in whichpreferred embodiments of the invention are illustrated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention identifies classes of novel compositions and novelmethods of using them, as well as novel methods of using classes ofknown compositions to kill anaerobic tumor cells and to enhance theeffectiveness of aerobic treatments for killing aerobic tumor cells.

The inventors have identified six categories of glycolytic inhibitorsthat can be used according to the present invention.

1. Analogs that Increase Tumor Targeting and Uptake:

(a) Lipophilic analogs of 2-deoxy-D-glucose.

In order to increase the uptake of 2-deoxyglucose into the inner core ofslow growing anaerobic cells, increases in lipophilicity aids inpenetration and therefore higher levels reaching these inner cells. Inpart, such analogs might also work as lipophilic prodrugs of2-deoxy-D-glucose.

Examples: Lipophilic analogs include derivatives of hydroxyl groups likeesters, ethers, phosphoesters, etc. Others include the removal of thehydroxyl group and replacement with halogens like fluorine or iodine, orwith thiol or thioalkyl groups.

(b) Liposome formulated 2-deoxy-glucose and its analogs (also inhibitorsof glycolysis such as oxamate and iodoacetic acid). Due to the abilityto change the lipophilic nature of the outer surface of liposomes andentrap drugs for better delivery to different tissue sites, liposomeshave previously been shown to increase tumor uptake of a variety ofdifferent drugs and molecules in experimental tumor models.

(c) Enzymatically cleavable derivatives of 2-deoxyglucose.

Examples of this class of compounds include Glucoronides with glycosidesat the C-1 position. The rationale of the invention is that the level ofthe enzyme glucoronidase has been shown to be higher in tumors than innormal tissues. Thus, by synthesizing 2-deoxyglucose in the form of aglucoronide its concentration at the tumor site will be naturallyenhanced by the increased levels of glucoronidases which will cleave itand deliver active 2-deoxyglucose specifically to the tumor.

2. Analogs of D-hexopyranoses:

For this disclosure the word “hexopyranoses” is used to cover allconfigurational isomers of monosaccharides modified at C-6 by removal(replacement) of 6-OH or by blocking it.

Such analogs will not be transformed by hexokinase or glucokinase toglucose-6-phosphate and can potentially block both enzymes.

Examples: 6-deoxy-D-glucose, 6-fluoro-D-glucose, and6-O-methyl-D-glucose.

3. Analogs Blocking Transformation from Glucose 6-phosphate to Fructose6-phosphate:

(a) Such transformation requires the presence of an hydroxyl group atC-2. Therefore, analogs without hydroxyl or having the hydroxyl properlyblocked easily undergo the first step of 6-O-phosphorylation byhexokinase or glucokinase but will not undergo isomerization byphosphoglucose isomerase. In fact such compounds can efficiently blockthe enzyme.

Examples: 2-deoxy-D-glucose itself and its analogs. Other useful analogsare 2-deoxy-2-halo-D-glucose, for example 2-fluoro- or 2-iodo-D-glucose.

(b) Another way to stop isomerization is modification at C-1.Replacement of hydroxyl by fluorine (glucosyl fluoride) or simpledeoxygenation to 1-deoxy-D-glucose.

4. Analogs Blocking the Aldolase A Cleavage, Thus Blocking Formation ofTrioses from Fructose 1,6-bisphosphate:

Such a process requires the presence of hydroxyl groups at C-3 and C-4.Thus, for example, 3-deoxy or 3-fluoro-D-glucose or 4-deoxy or4-fluoro-D-glucose can be transformed to 4-fluoro-D-fructose1,6-bisphosphate, which will not be cleaved by aldolase A but will blockit.

5. Blocking any of the Transformations of Glyceraldehyde:

Alterations are made at any step where phosphorylation is involved. Hereeither 2-fluoro or 3-fluoro-glyceraldehydes or glycerates can be used.If the phosphoester is important for blocking the enzyme, then, forexample, 3-fluoro-2-phosphoglycerate is used to block transformation tophosphoenolpyruvate

Also, phosphothioesters or other phosphor modified analogs can block thetransformations of glyceraldehyde.

6. The Pyruvate Structure:

Keeping in mind that oxamate is a good inhibitor of lactatedehydrogenase, compositions or structures of the present invention whichare efficient blockers of lactate dehydrogenase are included. An exampleis 2-fluoro-propionic acid or it salts; 2,2-difluoro-propionic acid.

Other examples are a pyruvate modified at C-3 such as 3-halo-pyruvate;3-halopropionic acid, 2-thiomethylacetic acid.

In addition to the proposed use of inhibitors of glycolysis assupplements to current chemotherapeutic and radiation treatment, therecent emergence of anti-angiogenic factors aimed at cutting off theblood supply to tumors and thus creating a more anaerobic environment,indicates that the present invention will likely have applicability tothis newly developing cancer therapy as well;

General Synthesis of Novel Chemicals:

The following section discloses the synthesis of analogs of glycolyticintermediates and determines their toxic potencies in ρ^(o) cells and intumor cells pretreated with Oxphos inhibitors.

According to the invention, a series of compounds has been rationallydesigned and prepared, that can block various steps of the glycolyticpathway, and which thus have an effect on cell models A, B, and C.

It is commonly believed that the three irreversible reactions ofglycolysis catalyzed by hexokinase, phospho-fructokinase, and pyruvatekinase are the controlling elements of this pathway. Thus, it wouldappear that the design and synthesis of inhibitors should focus on thesesteps. However, due to the complexity of interactions between glycolysisand other pathways it is likely that restricting the focus to thecontrolling elements is too naive. Thus, we will consider all reactionsin the glycolytic pathway as potential targets and design compoundsaccordingly.

Specifically, we have designed and synthesized the following, notingthat the number in parenthesis after each analog corresponds to thestructure illustrated in the following pages

Hexokinase Blockers.

6-Fluoro-D-glucose (1) and 6-thio-D-glucose (2) are synthesized. Removalof the hydroxyl group from the C-6 carbon in glucose and replacementwith fluorine (6-fluoro-D-glucose) and replacement with a thiol grouprespectively. 6-fluoro-D-glucose should prevent formation of glucose6-phosphate and inhibit hexokinase. 6-Thio-D-glucose (2) behavessimilarly to 6-fluoro-D-glucose (1) or may be transformed into a6-thiophosphate derivative. In either case, they effectively blockglycolysis.

Analogs Blocking Transformation from Glucose 6-phosphate to Fructose6-phosphate.

Transformation of glucose 6-phosphate to fructose 6-phosphate requires ahydroxyl group at C-2 in the glucose molecule. Therefore, analogslacking a hydroxyl in this position, or having a properly blockedhydroxyl might easily undergo 6-O-phosphorylation by hexokinase but willnot be able to undergo isomerization by phosphoglucose isomerase tofructose 6-phosphate. In fact, 2-dg is an example of such a glucoseanalog that efficiently stops glycolysis at the isomerization step.Other potentially useful C-2-modified analogs that can be prepared are2-bromo-D-glucose, 2-fluoro-D-glucose, and 2-iodo-D-glucose (4-5).

In addition, it should be possible to stop isomerization viamodification at C-1 or C-5. In the case of C-1, the hydroxyl at C-1 isreplaced with fluorine glucosyl fluoride (7) or with hydrogen to produce1-deoxy-D-glucose. In the case of C-5, sulfur replaces oxygen in thehexopyranose ring of glucose to obtain 5-thio-D-glucose (8).

Analogs Blocking Aldolase-catalyzed Cleavage.

In the next step of glycolysis, fructose-6-phosphate is transformed tofructose 1,6-biphosphate. Subsequently, fructose 1,6-biphosphateundergoes aldolase catalyzed cleavage to glyceraldehyde 3-phosphate anddihydroxyacetone phosphate. The cleavage reaction requires that fructose1,6-biphosphate contains hydroxyl groups at positions C-3 and C-4. Toexploit this requirement, we synthesize two glucose analogs modifiedwith fluorine atoms at these positions, i.e. C-3 (3-fluoro-D-glucose)(9)and C-4 (4-fluoro-D-glucose)(10). These analogs are expected to undergouninterrupted glycolysis to 3-fluor-fructose 1,6 biphsphate and4-fluoro-, fructose 1,6-bisphosphate; however, neither of them should beable to undergo aldolase-catalyzed cleavage to trioses. Increasedaccumulation of such compounds should stop glycolysis at this step.

Analogs Blocking Glyceraldehyde 3-phosphate Transformation to Pyruvate.

Steps that occur later in the glycolytic pathway i.e. fromglyceraldehyde 3-phosphate to pyruvate, are also targets for inhibitorsof glycolysis. The transformation of glyceraldehyde 3-phosphate to1,3-bisphosphoglycerate catalyzed by glyceraldehyde 3-phosphatedehydrogenase can be blocked by 1,1-difluoro-3-phosphate-glycerol (11)and 1,1-difluoro-glycerate (12) is synthesized. Furthermore, analogsmodified at C-2 are prepared with the rationale that substitution atthis position will affect the action of either one or all of thefollowing enzymes, glyceraldehyde 3-phosphate dehydrogenase,phosphoglycerate kinase, and phosphoglyceromutase. Examples of thesepotential glycolytic blockers are 2-fluoro-, 2-iodo-, 2-thio, or2-methoxy-glyceraldehydes (13-16) or glycerates (17-20).

Considering the next step in the glycolytic pathway C-3-substitutedtrioses will have inhibitory properties by virtue of being able to blockthe catalyzed step. The analogs are 3-fluoro-, 3,3-difluoro-, enolase3-iodo-, 3-carboxylo- and 3-thioglycerates (21-24).

Increasing the Lipophilicity of 2-dg

Lipophilic analogs of 2-dg act as prodrugs. Specifically, mono anddiesters of 2-dg are designed for increased uptake in tumor cells. Suchlipophilic esters enter the cell via passive diffusion instead ofrelying on glucose transporters and then are hydrolysed by esterases,leading to the release of 2-dg. Valerate, myristate, and palmitate areexamples of lipophilic derivatives of 2-dg (26-37). Specifically usefulare all four possible monoesters of 2-dg for each acid.

The structures of the inhibitors of the present invention are:

Diesters, however, are also useful. The pharmaco-kinetics of these monoand diester analogs of 2-dg, are different than that of the parentcompound.

Test Results on Type (B) ρ^(o) Cells.

When an osteosarcoma cell line (143B; Wild type in Table 1 below) wasexposed for prolonged periods to ethidium bromide a mutant completelydevoid of mitochondrial DNA ρ^(o) was isolated. As a consequence thismutant cell line, referred to as either ρ^(o) or cell line 206, cannotform intact respiratory chain complexes and therefore does not undergoelectron transport and or oxidative phosphorylation. These processes arenecessary for transmembrane proton pumping which creates anelectronegative membrane potential (Δψmt) in the matrix side of theinner mitochondrial membrane. Since Rho 123 is a fluorescent dye, whichdue to its chemical positive charge accumulates in mitochondria, it iscommonly used as an indicator of Δψmt. Thus, our results which show thatthe majority of ρ^(o) cells stained dimly, while wt cells stainedbrightly after a 10 min. exposure to Rho 123, indicate that ρ^(o) cellsdo indeed have an Δψmt, albeit reduced when compared to that in wtcells.

A mechanism by which ρ^(o) cells are able to generate a Δψmt, hasrecently been proposed to occur via reversal of ATP transport intomitochondria through the adenine nucleotide transporter (ANT). Thus, byexchanging ADP for ATP, the internal environment of mitochondria becomesmore electronegative. This process, however, does not appear to restorethe Δψmt, to its normal value.

Flow cytometric analysis was performed in order to quantitate thedifferences observed microscopically between wild type 143B (wt) andρ^(o) cells (designated as cell line 206) in their accumulation of Rho123. A four-fold decrease in Rho 123 retention in ρ^(o) cells ascompared to the parental wt has been observed. This data agrees withresults from other investigators working with these same cells (Bochetand Godinet, and Barrientos and Moraes), in which they estimate thatΔψmt of the ρ^(o) cells to be 3-4 times lower than wt.

To ensure that our accumulation results were not complicated byP-gp-mediated MDR, since Rho 123 is a known substrate for thistransporter, both cell types were examined for this gene by RT-PCR andwere found not to express it. An interesting observation which may havesignificance for clinical application of Rho 123 and verapamil fordetecting P-gp in freshly excised tumors or tumor samples from cancerpatients is that in these non-P-gp cell lines, verapamil increases theretention of Rho 123.

The ρ^(o) Cell Model as a Tool to Determine Whether Drugs Use FunctionalMitochondria as a Cytotoxic Target.

Drugs which depend on functional mitochondria for localization,accumulation or for free radical formation, should be less potent inρ^(o) than in equivalent wt cells. Thus, it is not surprising to findthat in growth inhibition studies, as shown in Table 1, ρ^(o) cells are50 and 6 times more resistant to Rho 123 and saffranin O (anothermitochondrial cationic dye) than wt cells, respectively.

TABLE 1 50% Growth Inhibition Dose (μg/ml) (CELLS) Drugs Wild type ρ°Ratio Rhodamine 123 1.0 50 50 Saffranin O 0.35 2 6 Doxorubicin 0.01 0.011 Vinblastine 0.00075 0.00075 1 Paclitaxel 0.05 0.05 1 2-deoxy-glucose1000 50 0.05

The differences between wt and ρ^(o) cells in their sensitivities tothese mitochondrial localizing drugs appears to be due to the lack offunctional mitochondria as a target, and the lowered Δψmt, in the ρ^(o)cell, which leads to reduced accumulation of Rho 123 and Saffranin O inthis latter cell type. Since Rho 123 acts as an uncoupler in isolatedmitochondria, and it is dependent on the strength of both Δψmt, andplasma Δψ for intracellular accumulation, it is not surprising to findρ^(o) cells to be more resistant than wt cells to this drug. This,however, does not imply that Rho 123, or other mitochondrial localizingdrugs kill the cell by inhibiting oxidative phosphorylation. It is notclear from our results whether a normally functioning cell with intactmitochondria will die if their mitochondria become uncoupled.

The fact that ρ^(o) cells (cell model (B)) are able to grow withoutundergoing oxidative phosphorylation demonstrates that blockage of thisprocess alone does not lead to cell death. It can be argued however,that ρ^(o) cells have been selected to survive without oxidativephosphorylation capability and therefore have developed other mechanismsfor this purpose. Thus, they are not equivalent to cells with normallyfunctioning mitochondria that are treated with uncouplers (cell model(A)).

Nevertheless, the increased accumulation of the positively-chargeddrugs, Rho 123 and saffranin O, in wt vs ρ^(o) cells, due to theincreased Δψmt, in the former cell type, most likely contributes totheir increased sensitivities to both drugs. In fact, as mentionedabove, Bochet and Godinet, and Barrientos and Moraes, estimate thatΔψmt, of these ρ^(o) cells to be 3-4 times lower than wt which agreeswith our results that show ρ^(o) cells retain 4 times less Rho 123 thanwt. Thus, by whatever mechanism Rho 123 ultimately kills a cell, theintracellular level of drug accumulation necessary for this actionappears to be facilitated by the strength of the Δψmt However, thepreliminary data we present with the cell model above, shows that whencells with normally functioning mitochondria, 143B or MCF-7, areco-treated with Rho 123 and glycolytic inhibitors, their survival iscompromised. This clearly indicates that mitochondrial agents whichinhibit Oxphos can hypersensitize tumor cells to glycolytic inhibitorsand that this combinative effect is indeed the mechanism by which theyinhibit cell growth and/or kill tumor cells.

In contrast, the nuclear localizing chemotherapeutic agent, Dox, isfound to be equitoxic in both cell lines of cell model (B), indicatingthat functional mitochondria are not an important site for its cytotoxicaction. Likewise, the tubulin-binding agents, paclitaxel and vinblastineare also found to be equitoxic in these cell lines. Moreover, asmentioned above with cell model (A), these drugs do not hypersensitizetumor cells to glycolytic inhibitors. Therefore, this data also acts asan important negative control to demonstrate that compounds which arenot known to preferentially accumulate in mitochondria or interact withits function, do not hypersensitize tumor cells to glycolyticinhibitors. In fact, this data further indicates that both cell models(A) and (B) are specific in testing whether inhibition of Oxphos leadsto hypersensitization to glycolytic inhibitors which results in growthinhibition and or cell death.

It should be noted here that the reason that ρ^(o) cells do not displayan MDR phenotype to the chemotherapeutic agents we have tested, eventhough they do not undergo Oxphos, is because they are growing rapidlyin vitro. This is quite different from the in vivo situation where acell in the middle of a tumor due to a number of reasons, isslow-growing and thus are resistant to most chemotherapeutic agentswhich target rapidly dividing cells. It is the hypoxic environmenthowever that leads to the anaerobic metabolism these inner core tumorcells undergo, which makes them selectively sensitive to glycolyticinhibitors. This is the essence of our discovery.

As predicted, ρ^(o) cells are more sensitive to 2-dg than wt cellsshowing an ID50 of 50 as compared to 1000 mg/ml for wt cells. Thus outof the six drugs presented in Table 1 above, 2-dg is the only one thatρ^(o) cells are hypersensitive to. Preliminarily we have found similardifferential sensitivity to oxamate and irdoacetate.

Thus, our data clearly support our working model that inhibition ofOxphos by either mitochondrial agents (cell model (A)) or by mutation inmitochondrial DNA, ρ^(o) cells, (cell model (B)) renders both of themhypersensitive to glycolytic inhibitors. The third part of our equation,cell model (C), is at the center of our studies, in that tumor cellsgrowing under hypoxic conditions are hypersensitive to inhibitors ofglycolysis. The present invention, based on the differences andsimilarities of our three cell models should lead to a new approach tochemotherapy.

One of the problems with successful treatment of solid tumors iseradicating all of the tumor cells including those that are morecentrally located. Due to their location, cancer cells at the inner coreof the tumor are less oxygenated and therefor rely more on anaerobicmetabolism in order to survive. In this condition they divide lessrapidly than the more aerobic tumor cells and thus are more resistant tothe standard chemotherapeutic agents which take advantage of rapidity ofreplication as a selective mode of toxicity. In addition, anaerobiccells are less able to produce free radicals when treated withchemotherapeutic agents or irradiation which utilize this part of thecell's capability in order to further potentiate its mode of action.Thus, anaerobiosis in these instances act as another component ofresistance to successful chemotherapeutic and radiation treatment.

It should be noted that a gradient of anaerobiosis exists within tumors,therefor it is expected that the more anaerobic the cell becomes themore susceptible it becomes to glycolytic inhibitors, such as2-deoxyglucose, oxamate and iodoacetate (the latter has been shown toblock glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step)and the analogs disclosed in this application. On the other hand, themore aerobic the cells become, the more sensitive they are toconventional chemotherapy and or radiation.

Therefore, we propose that glycolytic inhibitors, such as2-deoxyglucose, oxamate, iodoacetate and the analogs we specify here,significantly increase the efficacy of standard cancer chemotherapeuticand radiation regimens as well as new protocols emerging withanti-angiogenic agents by selectively killing anaerobic tumor cells whengiven in combination with the above mentioned therapies.

Specifically, we have discovered that 2-dg or glycolytic inhibitors ingeneral can be added to standard chemotherapy protocols that now existfor carcinomas and sarcomas, a few of which are listed below asrepresentative examples, i.e.:

Breast cancer: Cytoxan (a trademark for cyclophosphamide, marketed byBMS), Adriamycin (a trademark fordoxorubicin, marketed by Pharmacia),and 5FU, or Taxotere (a trademark for docetaxel, marketed by Aventis);

Sarcomas: Adriamycin I.V. and Cisplatin I.A. (intra arterial) orifosphamide, Adriamycin and DTIC (all I.V.);

Lung cancer: Navelbine (a trademark for vinorelbine) and cisplatin orTaxotere and carboplatin or gemcitabine and cisplatin;

Small Cell Lung cancer: VP-16 and cisplatin;

Head and Neck cancer: 5FU and cisplatin;

Colon cancer: 5FU and leukovorin and CPT-11 (irinotecan); and

Multi-drug resistant (MDR) tumors: MDR tumors will be treated with aglycolytic inhibitor and drugs active against MDR tumors. Example:combination of 2-dg with Annamycin. Such combinations will be especiallyuseful against breast cancer.

We also disclose treatment which combines blockers of oxphos withglycolytic inhibitors for general antitumor therapy. Treatment with theformer agents will convert aerobic tumor cells to anaerobic, thus makingthem hypersensitive to glycolytic inhibitors. Blockage of Oxphos can beaccomplished with agents that, in general, are positively-charged andare relatively lipophilic, examples of which are rhodamine 123,saffranin O, octylpyridium and others. In the case when tumor uptake isreduced by the presence of drug transporting proteins, such as P-gp orMRP (efflux proteins), this treatment will be used in combination withagents that block efflux, e.g. verapamil, cyclosporin.

Alternatively, lipophilic cationic compounds which inhibit oxidativephosphorylation in intact cells, but are not recognized by MDRtransporting systems, such as alkylguanidinium analogs, can be used inthe MDR+aerobic tumor cells to convert them to anaerobic cells andthereby render them hypersensitive to glycolytic inhibitors.

Moreover, glycolytic inhibitors are effective as single agent treatmentsfor tumors which are mostly anaerobic and because of their size andlocation cause discomfort and blockage of normal function, i.e. head andneck tumors. These inhibitors are useful in reducing tumor size whenadministered orally, IV, IP or directly into the tumor.

Another application of the glycolytic inhibitors of the presentinvention is in the treatment of bacterial infections which involveanaerobes, such as clostridia, bacteriodes, etc.

Detail Syntheses of Novel Compounds:

Synthesis of the novel compounds can be achieved by those of ordinaryskill in the art by understanding the following examples. Synthesis of3,4,6-Tri-O-aceryl-D-glucal can be performed by the scheme:

Compound 41 is then deacetylated to glucal 42, which is a keyintermediate for the invention. First, it is deoxygenated at C-2 as2-deoxy-glucose, but more importantly it contains hydroxyl groups thatdiffer in their reactivity. This allows differentiation during blockingreactions and selective preparation of acylated derivative of D-glucal.Specifically, D-glucal 42 contains a primary hydroxyl at C-6 which isthe most reactive in the molecule. Subsequently the secondary hydroxylat C-3, because of its allylic position, is more reactive than thesecondary hydroxyl at C-4.

Synthesis of D-glucal and its reactivity are illustrated by thefollowing scheme:

4-O-acylated derivative of 2-dg is prepared first. This is done by firstblocking hydroxyls at C-6 and C-3, which will leave hydroxyl at C-4 openfor acylation. The blocking groups at C-3 and C-6 should be selectivelyremoved in the presence of esters. Such properties are demonstrated bysilyl ethers, which are stable in basic but unstable in acidicconditions, whereas acyl groups appear to be stable in the latterconditions. Therefore, D-glucal 42 will first be silylated at C-3 andC-6 with sterically bulky t-butyldimethylchlorosilane (TBS) to 43leaving a free hydroxyl at C-4 ready for the acylation with acylchloride to 44 in the following scheme:

Compound 44 has a reactive double bond, which is reacted with silanol tosubstitute 2-deoxy-D-glucose 45 in the following scheme. Thiselectrophilic addition reaction requires the presence oftriphenylphospine hydrobromide. Compound 45 is then deprotectedselectively at C-1, C-3 and C-6, yielding the desired 4-O-acylatedanalogs 29-31.

Using similar known strategies the other compounds of the invention canbe prepared by those skilled in the art without undue experimentation,once the motivations to make these compounds, specifically the desire tomake glycolytic inhibitors for the purpose of killing anaerobic cells,have been learned from the present disclosure.

The Novel Method:

Another aspect of the invention is a method of inhibiting one or moresteps of the glycolytic pathway using an effective amount of one of moreglycolytic inhibitors or schemes, in the presence of anaerobic cells,specifically for the purpose of thereby killing the cells. The disclosedcompounds, used singly or in combination, have been shown to achievethis end. Other inhibitors and other inhibiting schemes may also beused, once the basic premises of the present invention are understood.

For example, known or new genetic manipulations to inhibit or block oneor more steps in the glycolytic pathway may be used to practice themethod of the invention.

For a better understanding of the pathway, and in particular the tensteps of the glycolytic pathway, any one or more of which may inspirethe use of inhibitors or schemes to thwart for practicing the presentinvention, the pathway is set forth as follows:

Reactions of glycolysis Step Reaction 1 Glucose + ATP → glucose6-phosphate + ADP + H⁺ 2 Glucose 6-phosphate

fructose 6-phosphate 3 Fructose 6-phosphate + ATP → fructose1,6-bisphosphate + ADP + H⁺ 4 Fructose 1,6-bisphosphate

dihydroxyacetone phosphate + glyceraldehyde 3-phosphate 5Dihydroxyacetone phosphate

glyceraldehyde 3-phosphate 6 Glyceraldehyde 3-phosphate + P_(i) + NAD⁺

1,3-bisphosphoglycerate + NADH + H⁺ 7 1,3-Bisphosphoglycerate + ADP

3-phosphoglycerate + ATP 8 3-Phosphoglycerate

2-phosphoglycerate 9 2-Phosphoglycerate

phosphoenolpyruvate + H₂O 10 Phosphoenolpyruvate + ADP + H⁺ → pyruvate +ATP Step Enzyme Type ΔG^(OP) ΔG 1 Hexokinase a −4.0 −8.0 2Phosphoglucose isomerase c +0.4 −0.6 3 Phosphofructokinase a −3.4 −5.3 4Aldolase e +5.7 −0.3 5 Triose phosphate isomerase c +1.8 +0.6 6Glyceraldehyde 3-phosphate f +1.5 −0.4 dehydrogenase 7 Phosphoglyceratekinase a −4.5 +0.3 8 Phosphoglycerate mutase b +1.1 +0.2 9 Enolase d+0.4 −0.8 10 Pyruvate kinase a −7.5 −4.0A Rapid Screening Method for Identifying Inhibitors of OxidativePhosphorylation:

Lactate (more commonly known as lactic acid) is an end product ofglycolysis that is activated when a cell cannot use oxygen as its finalelectron acceptor. This occurs when cells are metabolizinganaerobically. Thus, under these conditions, pyruvate, which is the lastend product of glycolysis during aerobic metabolism, is furthermetabolized to lactate. We demonstrated that agents such as rhodamine123 which inhibit oxidative phosphorylation in intact tumor cells(converting them from aerobic to anaerobic cells) hypersensitize them toinhibitors of glycolysis. This discovery created a need for a screeningmethod to identify agents which inhibit oxidative phosphorylation andthereby render a cell hypersensitive to glycolytic inhibitors.Consequently, we have developed a rapid and an inexpensive way to detectsuch agents by measuring increases in the production of lactate inliving cells.

We have found in our cell models A and B that measurement of the levelof lactate correlates with the degree of anaerobiosis. It can be seen inthe table below that in cell model B, ρo cells, which by nature of theirdeficiency in mitochondrial DNA cannot perform oxidative phosphorylationand are therefore metabolizing anaerobically, produce approximately 3times more lactate (15.4 μg/μg protein) than their aerobic parental cellcounterparts, 143b, (5.4 μg/μg protein). Similarly, in cell model A,(See table below) when the aerobically metabolizing cell, 143b, istreated with increasing doses of rhodamine 123, which should convert itto increasingly anaerobic growth, lactate is accordingly increased as afunction of increasing rhodamine 123 doses. In contrast,chemotherapeutic drugs which affect aerobic cells such as Doxorubicin(Dox), Vinblastine (Vbl) and Taxol (a trademark for paclitaxel) and donot hypersensitize cells to glycolytic inhibitors, show little or nosignificant changes in lactate production. We thus provide convincingevidence that increase in lactate is reflective of increases inanaerobic metabolism which can be induced by agents which inhibitmitochondrial oxidative phosphorylation. Such methodology provides apractical means for testing and or screening extensive libraries ofstructurally diverse compounds to identify inhibitors of oxidativephosphorylation as well as for obtaining new leads to design novelinhibitors of this fundamental cellular process.

TABLE 2 LACTATE PRODUCTION IN CELL MODELS A & B DOSE LACTATE CELL LINEDRUG (μg/ml) (μg/μg protein) ρo Control 0 15.4 +/− 0.3  143b Control 05.4 +/− 0.1 143b Rho 123 0.25 9.3 +/− 0.4 143b Rho 123 0.50 13.2 +/−0.2  143b Rho 123 1.0 14.6 +/− 2.3  143b Dox 0.005 5.1 +/− 0.4 143b Dox0.01 5.5 +/− 0.7 143b Vbl 0.0005 5.6 +/− 0.6 143b Vbl 0.001 6.3 +/− 0.9143b Vbl 0.002 5.8 +/− 0.3 143b Taxol 0.0005 5.6 +/− 0.7 143b Taxol0.0010 6.1 +/− 0.7 143b Taxol 0.0020 6.6 +/− 0.3

We have thus also demonstrated as part of this invention, that agentssuch as rhodamine 123 which inhibit oxidative phosphorylation in intacttumor cells, hypersensitize them to inhibitors of glycolysis byaccelerating anaerobic metabolism.

Weight Loss Method:

Based on the principle of this discovery, we claim the extension of theuse of this concept as a novel approach for weight control as follows:

Since anaerobic metabolizing cells take up and use glucose more rapidlythan aerobic metabolizing cells, we propose to add small amounts ofagents which localize in mitochondria and slow down oxidativephosphorylation. Thus, most cells in the body would have increaseduptake and utilization of glucose, thereby lowering blood sugar levels.Under these conditions, in order to compensate for reduced glucoselevels in the blood, gluconeogenesis would be increased by the liver.Gluconeogenesis is a process whereby glucose is synthesized by breakdownof fats. Thus, our treatment effectively increases the breakdown andutilization of stored fat. Currently, intense energetic exerciseaccomplishes the same thing most likely by a similar mechanism.

While specific embodiments of the invention have been shown anddescribed in detail to illustrate the application of the principles ofthe invention, it will be understood that the invention may be embodiedotherwise without departing from such principles.

1. A method of treating cancer in a human, said method comprising orallyadministering an effective amount of a pharmaceutical compositioncomprising 2-Deoxy-D-glucose in combination with a cancer chemotherapyprotocol, subject to the limitation that said protocol does not includeadministration of an inhibitor of oxidative phosphorylation and furthersubject to the limitation that said administration is in the absence ofradiation therapy.
 2. A method of killing cancer cells in a human,wherein said cancer cells include cancer cells that create ATP forenergy anaerobically and cancer cells that aerobically generate ATP,said method comprising administering a cancer chemotherapy protocol,said protocol not including administration of an inhibitor of oxidativephosphorylation in combination with orally administering a glycolvticinhibitor capable of inhibiting at least one step of the glycolyticpathway, wherein said therapy is administered without radiation therapy,and said glycolytic inhibitor is of 2-Deoxy-D-glucose.
 3. The methodaccording to claim 2, wherein the at least one compound of the cancerchemotherapy protocol is selected from the group consisting ofcyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, cisplatin,ifosphamide, DTIC, vinorelbine, carboplatin, gemoitabine, VP-16,leukovorin, an antiangiogenic agent, and irinotecan.
 4. A method fortreating a human with a carcinoma or sarcoma that contains bothaerobically and anaerobically metabolizing cells, said method comprisingthe steps of: (a) administering a chemotherapy protocol, said protocolnot including administration of an inhibitor of oxidativephosphorylation, for killing at least some of the aerobicallymetabolizing cells; and, in the absence of radiation therapy, (b) orallyadministering 2-Deoxy-D-glucose for killing at least some of theanaerobically metabolizing cells.
 5. The method according to claim 4,wherein the chemotherapy protocol includes administering at least one ofcyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, cisplatin,ifosphamide, DTIC, vinorelbine, carboplatin, gemcitabine, VP-16,leukovorin, an anti-angiogenic agent, and irinotecan.
 6. A method fortreating a human with a carcinoma or sarcoma that contains bothaerobically and anaerobically metabolizing cells, said method comprisingthe steps of: (a) administering a chemotherapy protocol wherein thechemotherapy protocol includes administering an anti-angiogenic agent,and said protocol not including administration of an inhibitor ofoxidative phosphorylation, for killing at least some of the aerobicallymetabolizing cells; and, in the absence of radiation therapy, (b) orallyadministering 2-Deoxy-D-glucose for killing at least some of theanaerobically metabolizing cells.
 7. A method of killing cancer cells ina human, wherein said cancer cells include cancer cells that create ATPfor energy anaerobically and cancer cells that aerobically generate ATP,said method comprising administering in the absence of radiationtherapy, an anti-angiogenic agent in combination with orallyadministering a glycolytic inhibitor capable of inhibiting at least onestep of the glycolytic pathway, wherein said glycolytic inhibitor is2-Deoxy-D-glucaose.